Faithful inheritance of mitochondrial DNA (mtDNA) is essential for cellular respiration and thus for most eukaryotic life. However, pathways that control mtDNA maintenance and segregation in mammalian cells are not well understood. The goal of this work is to understand the molecular mechanisms underlying the regulation of mammalian mtDNA. Once this information is known, it will be possible to identify factors that contribute to the segregation of mtDNA haplotypes that impact human health. Specifically, this proposal will address how mtDNA-protein complexes, termed nucleoids, behave in mammalian cells. In Aim 1, super-resolution and confocal microscopy of nucleoid complexes coupled with genetic approaches to deplete and modify complex components will be employed to investigate the intracellular basis of nucleoid segregation and test the involvement of candidate effector proteins. Aim 2 will address how the nucleoid is organized or integrated with cellular signaling pathways. This will be accomplished by systematically mapping physical and genetic interactions of the mammalian mtDNA nucleoid complex. These experiments will provide insight into the physical and functional organization of the heritable unit of mtDNA and effectively map the integrated 'interactome' of the nucleoid structure.